[Show abstract][Hide abstract] ABSTRACT: Meningiomas are the second most common primary tumors of the central nervous system. Meningiomas at the cranial base pose technical challenges and result in increased morbidity. To investigate the molecular mechanisms of meningioma formation, the expression profiles of 12 000 genes from meningiomas and dural specimens were compared.
Ribonucleic acid from 6 meningiomas (World Health Organization Grade I) and 4 dural specimens was profiled using U95A GeneChips (Affymetrix, Inc., Santa Clara, CA). Expression profiles of the 2 groups were compared using dChip and Data Mining Tool software packages (Affymetrix, Inc.) to identify differentially expressed genes. Down-regulation of a differentially expressed tumor suppressor gene, deleted in liver cancer 1 (DLC1), was verified by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Function and methylation of DLC1 were assessed by ectopic expression in 5 primary cultures, demethylation assay using 5-aza-2'-deoxycytidine, and methylation-specific polymerase chain reaction in 4 meningioma samples.
Gene expression profiling revealed up-regulation of 5 genes (fibroblast growth factor 9, gibbon leukemia virus receptor 2, cyclin D1, eukaryotic translation initiation factor 5A, and 28S ribosomal ribonucleic acid) and down-regulation of 35 genes, including DLC1, in meningiomas. The down-regulation of DLC1 in meningiomas was confirmed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining. Transfection of DLC1 complementary deoxyribonucleic acid into primary cultures of 5 meningiomas resulted in decreased replication. Although demethylation decreased meningioma cell growth rates in vitro, methylation-specific polymerase chain reaction did not detect DLC1 promoter methylation.
The results suggest that DLC1 may function as a tumor suppressor gene in meningiomas. Furthermore, DLC1 promoter methylation does not appear to be responsible for the decreased DLC1 expression in these tumors.
[Show abstract][Hide abstract] ABSTRACT: Promotion of the repair and regeneration of damaged adult neurons is a major goal of neurological science. In this study, the effects of G protein-coupled receptor kinase interacting protein 1 (GIT1) overexpression in human neuron cells were tested in human neuronal cells by using an adenoviral vector.
A recombinant GIT1 and enhanced green fluorescent protein (EGFP) adenoviral vector (AdGIT1) was created by using a standard viral construction procedure. Human neuronal (NT2N) cells, which had been derived from an NT2 human teratocarcinoma cell line, were used in this experiment. Immunocytochemical methods were applied to identify NT2N cells with neural features and to probe the relationship among signaling proteins. Several biological activities were assessed, including neural spine formation, cell migration, and the levels of expression of growth-associated protein-43 (GAP-43) and active Cdc42. The number of cells with spine formation and the number of migrated cells were significantly higher in the AdGIT1-treated group of NT2N cells than in untreated (control) NT2N cells or in AdEGFP-treated NT2N cells. The levels of GAP-43 and active Cdc42 expression were significantly higher in the AdGITl-treated group than that in the other two cell groups.
The results of this study demonstrate that GIT1 overexpression has the potential to promote neural spine formation and cell migration in human neuronal cells. At the same time, the increased level of GAP-43 in GIT1-overexpressed cells indicates that GIT1 may have the potential to improve growth and regeneration of damaged axons. The GIT1-beta-PIX-Cdc42-PAK pathway may play an important role in neuronal outgrowth.
Journal of Neurosurgery 08/2006; 105(1):103-10. DOI:10.3171/jns.2006.105.1.103 · 3.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteogenic potentials of some recombinant human bone morphogenetic protein (BMP) first-generation adenoviral vectors (ADhBMPs) are significantly limited in immunocompetent animals. It is unclear what role expression of viral proteins and foreign proteins transduced by adenoviral vectors play in the host immune response and in ectopic bone formation. In this study two sets of experiments were designed and performed. First, rat BMP6 cDNA were amplified, sequenced, and recombined in first-generation adenoviral vector (ADrBMP6). A comparison of human and rat BMP6 adenoviral vectors demonstrated identical osteogenic activities in both immunodeficient and immunocompetent rats. Second, the activities of recombinant human BMP6 in E1- (ADhBMP6) and [E1-,E2b-] ( [E1-,E2b-]ADGFP&hBMP6, and [E1-,E2b-]ADhBMP6) adenoviral vectors were compared in both in vitro and in vivo models. Similar activities of these two generations of BMP adenoviral vectors were found in all models. These results indicate that the amount of viral gene expression and the source of the BMP cDNA are not major factors in the interruption of osteogenic potentials of recombinant BMP6 adenoviral vectors in immunocompetent animals.
International journal of medical sciences 06/2006; 3(3):97-105. · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The osteogenic potential of AAV5hBMP6 was compared with that of ADhBMP6 in immunodeficient and immunocompetent rats. AAV5hBMP6 (2.3 x 10(12) particles) and ADhBMP6 (5 x 10(7) PFU) elicited viral antibody production in immunocompetent rats. Among rats that received AAV5hBMP6, the earliest time points at which the bone was visible under CT scanner were 30 days in 2-month-old Sprague-Dawley (SD) rats and 60 days in 18-month-old SD rats. The mean volumes of ectopic bone 90 days after viral injection were 0.31 +/- 0.14 cm(3) in athymic nude rats, 0.64 +/- 0.12 cm(3) in 2-month-old SD rats, and 0.21 +/- 0.10 cm(3) in 18-month-old SD rats. In contrast, among rats that received ADhBMP6, the earliest time points to observe the bone formation by CT scan were 15 days in 2-month-old rats and no bone formation in 18-month-old SD rats. The mean volumes of ectopic bone were 4.17 +/- 0.05 cm(3) in athymic nude rats and 0.06 +/- 0.03 cm(3) in 2-month-old SD rats. Although both types of viruses induced an immune response in immunocompetent animals, this response played different roles in the process of bone formation induced by the BMP6 vectors.
[Show abstract][Hide abstract] ABSTRACT: Mature mammalian neurons, except for olfactory neurons, are incapable of cell division. Promoting the regeneration of degenerated adult neurons is a major goal in neurological science. The human teratocarcinoma cell line (NTera 2 or NT2) is capable of neuronal differentiation following treatment with retinoic acid. These neurons (NT2N cells) have a stable neuronal phenotype. The effect of GIT1 overexpression in human NT2N cells was evaluated using an adenoviral vector. A recombinant GIT1 and GFP adenoviral vector (AdEGFP-GIT1) was constructed. The biologic activities of GIT1 were assessed according to the induction of neuronal spine formation, cell migration, and the amounts of GAP-43 and active Cdc42. Cells treated with AdEGFP-GIT1 were compared to untreated cells and to cells treated with AdEGFP. NT2N cells in the AdEGFP-GIT1 group demonstrated more spine formation and higher numbers of migrating cells than that either cells treated with AdEGFP or untreated NT2N cells. GAP-43 and active Cdc42 were quantified by dot ELISA method. The results showed that the amount of GAP-43 and active Cdc42 in the AdEGFP-GIT1 group was much higher than in AdEGFP and untreated NT2N groups. In conclusion, GIT1 overexpression promotes spine formation, migration, and expression of the growth factor GAP-43 and active Cdc42. The GIT1/β-PIX/Cdc42/PAK pathway may play an important role in the biological activities of neurons. GIT1 also stimulates GAP-43 expression which may help promote regeneration of damaged axons and formation of new synapses. Therefore, GIT1 may have potential in the treatment of nerve injury.
[Show abstract][Hide abstract] ABSTRACT: Based on previous gene therapy studies in bone formation induced by BMP adenoviral vectors, the osteogenic potentials of different BMP adenoviral vectors (ADhBMP-2, -4, -6, -7, and -9) are very high in immunodeficient animals but vary significantly among immunocompetent animals. We have demonstrated that the amounts of bone formation in immunocompetent animals were not significantly affected by use of different generations of adenoviral vectors and different species of BMPs. The similar protein expression curves between immunodeficient and immunocompetent rats indicated that reduced amount of BMP expression was not the main factor limiting the bone formation. Nevertheless, some components of sera may specifically block the signal transduction pathways of particular BMPs, thus interrupting bone formation. We use the term “bone formation inhibitors (BFIs)” to describe these immune factors to distinguish them from “BMP antagonists”, which are mainly secreted by bone tissue. A mouse skeletal muscle myoblast cell line (C2C12) was used as a model. Reduction of alkaline phosphatase (AP) expression was used as an index to evaluate the inhibiting activity of serum. The distribution of BFIs among different animal species and strains was investigated. Sera from rabbit, rat, and mice were tested. Sera from two strains of rabbit included 14 normal control sera and five adenovirus-vaccinated sera. Most rabbit sera did not exhibit significant inhibiting activity on cell response to any BMP. Only one serum from adenovirus-vaccinated rabbit displayed inhibiting activity on cell response to BMP6. Two sera from NOD SCID mice did not show any inhibiting activity on cell response to BMP2, -6 and -9. Forty-four sera obtained from athymic nude, SD, and Wistar rats were tested. From athymic nude rats we obtained five normal and seven adenovirus-vaccinated sera. All the rat sera inhibited response of cells to BMP6. Two sera obtained from adenovirus (ADLUC)-vaccinated athymic nude rats 16 months after the vaccination inhibited cell response to BMP2, -6, and -9. Four sera from untreated Wistar rats demonstrated inhibiting activities to all three BMPs. Sera from 13 normal, and 15 adenovirus-vaccinated SD rats, all displayed inhibiting activity to BMP6. Some sera also showed inhibition of cell response to BMP2 and -9. The results indicate that BFIs are widely distributed among different animal species and strains. Adenovirus-vaccinated sera may have higher titers than normal sera. This finding will provide a new point of view to assess the relationship between the immune system and BMPs.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to see if immunosuppression achieved using local application of cyclosporine A (Cs. A) or CD4 and CD8 antibodies would improve bone formation following intramuscular injections of human BMP-4 and BMP-9 adenoviral vectors (ADhBMP4 and ADhBMP9) in Sprague-Dawley rats. Cs. A was injected into the thigh muscle. After 2 days, ADhBMP4, ADhBMP9, and the antibodies were separately injected into the left and right rear legs. At this time, the number of CD4+/CD3+ cells was significantly lower and the number of CD8+/CD3+ cells higher in the Cs. A group than in the control group (P < 0.01). The total number of white blood cells 3 days following injection of CD4 and CD8 antibodies was significantly lower than that before the injection (P < 0.01). At 4 weeks after the viral and antibody injections, mean bone volumes at the ADhBMP9 treatment sites were 0.29 +/- 0.01 cm3 in the viral control group, 0.17 +/- 0.03 cm3 in the Cs. A-ADhBMPs group, and 0.59 +/- 0.07 cm3 in the antibodies-ADhBMPs group. ADhBMP4 did not induce new bone formation in any group. This study demonstrates that local immunomodulation may improve the osteogenic potential of bone morphogenetic protein gene therapy in the clinical setting.
[Show abstract][Hide abstract] ABSTRACT: Luciferase optical imaging provides a novel method to monitor transgene expression in small living animals. As the genetic and immunological heritages of particular animals significantly affect the expression of adenovirus-delivered transgenes, it is essential to know the expression patterns specific to athymic nude and Sprague-Dawley rats, two strains commonly used in rodent models. In this study we set out to determine these patterns. At the same time, we tested luciferase optical imaging in a larger animal, the rabbit.
A recombinant luciferase adenoviral vector was injected subcutaneously or intramuscularly into athymic nude rats, Sprague-Dawley rats, and Dutch Belted rabbits. The luciferase expression was assessed using a cooled charge-coupled device.
The luminescent signal was capable of passing through at least 1.3 cm of muscle tissue and proved to be much stronger when luciferin was delivered via a local injection than by an intraperitoneal injection. Although the types of immune cells differed between immunodeficient and immunocompetent rats, similar amounts and patterns of luciferase expression were observed in the musculature in two rat strains during the 1st month after a viral intramuscular injection. The duration of luciferase expression was longer than 15 months in athymic nude rats, 9 months in Sprague-Dawley rats, and 6 months in rabbits following a direct viral injection.
Luciferase expression after adenoviral gene delivery can persist for longer than 6 months, even in immunocompetent animals. Live imaging of luciferase expression can be performed not only in small animals, but also in larger animals such as rabbits.
The Journal of Gene Medicine 06/2005; 7(6):792-802. DOI:10.1002/jgm.720 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adeno-associated virus (AAV) and adenovirus (AD) are the two most commonly viral vectors in gene therapy. Both vectors have their own advantages and limitations. In this study, a human bone morphogentic protein 6 (hBMP6) cDNA under control of an immediate CMV promoter was separately inserted into AAV type 5 (AAVhBMP6) and human adenovirus type 5 (ADhBMP6) vectors. Since no a reasonable viral unit could be used to give definite dose equivalence between these two viral vectors, the viral doses were chosen based on our previous experiments with these two vectors. The criterion to choose the viral dose was the amount of viral vector having the potential to induce bone formation in both athymic nude (AN) and Sprague-Dawley (SD) rats. Two-month-old AN and SD (15 of each strain) and 18-month-old SD (3 animals) rats were used in this experiment. Five young rats from each strain were directly injected with AAVhBMP6 (7 × 1012 particles/350 μl) or ADhBMP6 (5 ×107 PFU/350 μl) into the left thigh separately. Three old SD rats were injected with the above amount of AAVhBMP6 in the left thigh and ADhBMP6 in the right thigh. The percentages of CD4, CD8, CD11b, and CD45RA cells were measured 3 days later by using flow cytometry. The animals were scanned with CT on Days 15, 30, 60, and 90 after viral injection. The AN rats had significantly lower percentages of CD4+ (27.7%) and CD8+ (16%) than same age SD rats (CD4+ 43.4% and CD8+ 31.5%), but higher percentages of CD11b+ (38.7%) than SD rats (CD11b+ 15.8%). Most of the CD4+ and CD8+ cells in athymic nude rats were double stained with CD11b+. That indicates that these CD4+ and CD8+ cells in AN rats are macrophage cells, rather than true T cells. Two-month-old SD rats had the lower percentages of CD8+ (16%) and CD11b+ (15.8%) than 18-month-old SD rats (CD8+ 39.5% and CD11b+ 32.7%). The percentages of all cell types were not significantly different among different groups in the same age and strain of rats. The average amounts of bone, calculated only among rats that had the bone formation in each group, on Days 15, 30, 60, and 90 were the following: 0, 0.12, 0.25, and 0.23 cm3 for AAVhBMP6 (3/4 rats have bone, the same as following) and 2.52, 3.96, 3.65, and 3.98 cm3 for ADhBMP6 (5/5) in AN rats; 0, 0.15, 0.3, and 0.28 cm3 for AAVhBMP6 (5/5) and 0.07, 0.06, 0.07, and 0.08 cm3 for ADhBMP6 (1/5) in 2-month-old SD rats, and 0, 0.15, 0.20, and 0.18 cm3 for AAVhBMP6 (3/3) and 0 for ADhBMP6 (0/3) in 18-month-old SD rats. In addition, the viral concentration or volume may also affect the amount of bone formation because the same amount of ADhBMP6 in 50 μl induced 1.2 cm3 in AN (5/5) and 0.12 cm3 in SD (4/5) rats. These results indicate that AAVhBMP6 has the potential to induce ectopic bone formation in both immunodefective and immunocompetent animals but takes longer time to form bone than ADhBMP6 in both rat strains. Interestingly, the amount of bone induced by AAVhBMP6 is smaller than ADhBMP6 in athymic nude rats, but is larger than ADhBMP6 in SD rats. Similar sizes of bone induced by AAVhBMP6 are observed in both rat strains. Eighteen-month-old rats still have the ability to form the ectopic bone.
[Show abstract][Hide abstract] ABSTRACT: Although adenoviral vectors have a very broad range and high efficiency of cell transduction both in vitro and in vivo, the uses of adenoviral vectors have been significantly limited in immunocompetent animals. We tested whether the amount of protein expression was the major factor determining the foreign gene functions. The recombinant adenoviral vectors of human bone morphogentic protein 4 and luciferase were used to answer this question. Direct viral injection and ex vivo methods were chosen in immunodefective athymic nude (AN) and immunocompetent SD rats. On Day 0, the primary rat bone marrow cells were infected with either ADhBMP4 or ADLUC at 107 PFU/flask or PBS. At the same time, five AN and SD rats were given a direct injection of either ADhBMP4 or ADLUC (107 PFU/100 μl) into the right thigh. On Day 1, both control and virally infected cells were injected into the left thigh with 2 × 106 cells/100 μl/flask. The rats underwent imaging performed with a cooled CCD before and after an intraperitoneal luciferin injection on Days 1 and 7. On Day 1, the densities of luminescent signals (×107 photons/sec) were the following: 1.83 ± 0.56 for SD rats with direct ADLUC injection; 3.73 ± 1.15 for SD rats with ex vivo ADLUC injection; 5.01 ± 1.91 for AN rats with direct ADLUC injection; and 11.9 ± 3.35 for AN rats with ex vivo ADLUC injection. The luminescent signals in the control groups were the same as background. By Day 7, the percentages of luminescence densities in ADLUC groups (compared with the same site on Day 1) were the following: 14.1% for SD rats with direct ADLUC injection; 1.06% for SD rats with ex vivo ADLUC injection; 25.4% for AN rats with direct ADLUC injection; and 0.8% for nude rats with ex vivo ADLUC injection. The animals were scanned with CT 1 month after injection. The bone volumes (cm3) of ADhBMP4 induced were 0.85 ± 0.08 for direct injection and 0.15 ± 0.03 for ex vivo in AN rats, and no bone for direct injection and 0.12 ± 0.02 for ex vivo in SD rats. No bone was observed at sites receiving injections of control cells or ADLUC. These data demonstrate that AN and SD rats display similar expression profiles of foreign gene expression delivered with adenoviral vectors, regardless of whether direct viral injection or the ex vivo method is used. As we know the amount of new bone is dose dependent in AN rats, and the average amount of bone induced by hBMP4 is smaller when the ex vivo method is employed than when direct viral injection is used. This result reflects the lower level of hBMP4 expression using the ex vivo method than directly viral injection. However, in SD rats, although hBMP4 expression may be lower after the ex vivo injection than after directly viral injection, the ex vivo ADhBMP4 delivery induces bone formation. These results indicate that the amount of BMP expression is not the major factor affecting the amount of new bone in SD rats. The major factors affecting the osteogenic potentials of ADhBMPs in SD rats may be some immune factors, which may have higher titer in direct adenoviral injection comparing to the ex vivo delivery.
[Show abstract][Hide abstract] ABSTRACT: Different animal strains have different genetic backgrounds that influence their physiological function and pathological process. The differences in genetic background may affect the efficiency of adenoviral infection and target gene expression and further cause different gene therapy results when target genes are delivered with adenoviral vectors. In this study, ectopic bone was not seen in ADCMVBMP4 injection sites, but was formed in ADCMVBMP9 injection sites in all rat strains. The mean volumes of bone induced with ADCMVBMP9 were 0.87 +/- 0.2 cm3 in Wistar, 0.26 +/- 0.1 cm3 in Long-Evans, 0.34 +/- 0.2 cm3 in Sprague-Dawley, 0.44 +/- 0.1 cm3 in ACI, 0.66 +/- 0.2 cm3 in PVG, and 0.58 +/- 0.1 cm3 in Fischer 344 rats. This indicates that ADCMVBMP9 has different bone formation potentials in different immunocompetent rat strains (P = 0.02). The basic levels of CD4+ and CD8+ T cells in blood before viral infection and titers of adenoviral neutralizing antibodies 30 days post-viral infection were significantly different among rat strains (P < 0.01). The efficiencies of target gene expression delivered with adenovirus were also significantly different in primary muscle cell cultures from different rat strains (P < 0.01). The different osteogenic potentials of ADCMVBMP9 among rat strains may be, in part, due to the differences in immune factors and target gene expression efficiency in muscle tissue.
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic protein (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of bone pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate bone formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague-Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 x 10(5) PFU/well of BMP viral vector was 4890 x 10(-12) U/well for ADCMVBMP-9, 302 x 10(-12) U/well for ADCMVBMP-4, 220 x 10(-12) U/well for ADCMVBMP-6, 45 x 10(-12) U/well for ADCMVBMP-2, and 0.43 x 10(-12) U/well for ADCMVBMP-7. The average volume of new bone induced by 10(7) PFU of BMP vector in athymic nude rats was 0.37+/-0.03 cm(3) for ADCMVBMP-2, 0.89+/-0.07 cm(3) for ADCMVBMP-4, 1.02+/-0.07 cm(3) for ADCMVBMP-6, 0.24+/-0.05 cm(3) for ADCMVBMP-7, and 0.63+/-0.07 cm(3) for ADCMVBMP-9. In immunocompetent Sprague-Dawley rats, no bone formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of 10(8) PFU induced 0.10+/-0.03 cm(3) of new bone, whereas ADCMVBMP-9 at a lower viral dose of 10(7) PFU induced more bone, with an average volume of 0.29+/-0.01 cm(3).
[Show abstract][Hide abstract] ABSTRACT: Although recombinant first-generation BMP adenoviruses can induce ectopic bone formation in immune deficient animals, the osteoinductive activity of these BMP vectors is reduced in immune competent animals. Helper-dependent adenoviral vectors have been developed to decrease the immune response and, therefore, increase gene expression in immune competent animals compared with first-generation vectors. In the present study, the osteoinductive activity of a helper-dependent GFP and BMP-9 adenoviral vector (ADGBMP9) was evaluated in vitro and in vivo.
Initially, purified ADGBMP9 was used to transduce human mesenchymal stem cells (hMSCs) and the alkaline phosphatase activity was determined as a measure of osteogenic activity. The vector was then injected into the thigh muscle of athymic nude and Sprague-Dawley rats, and CT scans and histology were subsequently used to assess bone formation.
In vitro, ADGBMP9 was capable of inducing alkaline phosphatase expression in hMSCs. In vivo in athymic nude and Sprague Dawley rats, ADGBMP9 initiated the process of bone formation 3 days after percutaneous injection into the thigh musculature. The rats demonstrated intramuscular ectopic ossification in CT scans as early as day 9 post viral injection and ultimately formed significant amounts of ectopic bone. Histologically, the induced bone was formed via normal endochondral mechanisms.
A helper-dependent adenoviral vector containing the BMP-9 and GFP genes has significant osteoinductive activity in both athymic nude and immune competent rats. Additional direct and ex vivo BMP gene therapy studies are required to assess the vector's activity in more animal models.
The Journal of Gene Medicine 09/2003; 5(9):748-56. DOI:10.1002/jgm.412 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was undertaken to determine whether ex vivo bone morphogenetic protein-9 (BMP-9) gene therapy using human mesenchymal stem cells (hMSCs) can induce endochondral bone formation in athymic nude rats. An in vitro study was initially performed on hMSCs to evaluate morphological changes and osteoblastic differentiation induced by replication-defective adenovirus type 5 with the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or beta-galactosidase (Ad-beta-gal) gene. In vivo, athymic nude rats received an injection (10(6) hMSCs transduced with recombinant adenovirus at 50 PFU/cell) into the anterior thigh musculature: Ad-BMP-9 on the left and Ad-beta-gal (control) on the right. Computed tomography scans and histological analysis were obtained 7, 14, 28, 42, 56, and 84 days postinjection. In vitro, human mesenchymal stem cells treated with Ad-BMP-9 (50 PFU/cell) showed signs of differentiation, whereas hMSCs treated with 250 and 1250 PFU/cell showed cytotoxicity. In vivo, computed tomography and histological analysis clearly demonstrated ectopic bone at hMSC/Ad-BMP-9 treatment sites, whereas the hMSC/Ad-beta-gal treatment sites showed no evidence of osteogenesis. None of the animals showed clinical evidence of toxicity. Ex vivo gene therapy with hMSC/BMP-9 may be efficacious for promoting bone formation for a variety of bone pathologies and certainly warrants further investigations.
[Show abstract][Hide abstract] ABSTRACT: In vitro experiments are performed on a daily basis to study the molecular biology of tumors. For some benign tumors, however, established cell lines are not always available, or it may not be feasible to establish new ones. In such cases, primary cultures are used to perform in vitro experiments. Gene expression profiles in vitro differ from those in vivo, but information as to which genes have significantly altered levels is limited. In this study, gene expression profiles of meningiomas in primary cultures and frozen tumors were compared.
Affymetrix U95A chips were applied to three sets of meningiomas. For each tumor, the gene expression profiles in frozen specimens (Fr) and primary cultures from the same tumor at Pass 5 (P5) and Pass 10 (P10) were compared. A paired t test (P < 0.025) was applied between Fr and P5, and then between Fr and P10. Genes that demonstrated significantly different expression levels in both comparisons were then identified. The expression levels for a subset of these genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction.
Among 12,000 genes examined, up-regulation of 51 genes by fivefold or more and down-regulation of 19 genes by twofold or more was found in primary cultures (P5 and P10) compared with the corresponding Fr. Up-regulation of genes encoding for extracellular matrix, cytoskeleton, and cell surface receptors was particularly notable.
Gene expression of tissue-cultured meningiomas and in situ meningiomas is significantly different for a large number of genes. Therefore, gene expression and therapeutic studies on cultured meningiomas need to be interpreted with caution.
[Show abstract][Hide abstract] ABSTRACT: Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat.
Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the beta-galactosidase (Ad-beta-gal) gene. Ad-beta-gal served as the control. Each animal received a percutaneous, paraspinal injection of 10(6) hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-beta-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically.
Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-beta-gal-transduced hMSCs showed no evidence of osteogenesis.
The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic proteins (BMPs) delivered on scaffolds can induce ectopic bone formation after subcutaneous injection. Adenoviral vectors (Ad) carrying BMP2, BMP7, and BMP9 cDNAs have been shown to produce bone through endochondral ossification. The present study was performed to elucidate the histological events leading to ectopic ossification for two novel first-generation adenoviral constructs encoding BMPs, AdBMP4 and AdBMP6. In vitro, the viral constructs produced and secreted the mature BMP4 and BMP6 proteins. In vivo, the calf muscles of athymic nude rats were injected with AdBMP4, AdBMP6, AdBMP2, or AdlacZ. Rats were sacrificed 3, 6, 9, 16, 21, 60, and 90 days postinjection. Whereas AdBMP4 produced ectopic bone through mechanisms similar to endochondral ossification, AdBMP6 seemed to induce bone by way of mechanisms similar to both intramembranous and endochondral ossification pathways. At the relatively low vector dose used in this study, AdBMP2 caused an initial recruitment of primitive mesenchymal cells, without further development to bone. From computed tomographic analysis, AdBMP6 produced the most rapid tissue calcification. The ultimate density of ectopic bone formed by AdBMP4 and AdBMP6 was comparable. The current study demonstrates that AdBMP4 and AdBMP6 are more potent than the prototypical osteogenic adenoviral vector AdBMP2 and seem to induce ectopic bone by different mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Survivin is an inhibitor of apoptosis protein that blocks apoptosis by binding to caspases-3 and -7. It is highly expressed in less-differentiated embryonic cells and rapidly dividing tumors, but not in terminally differentiated adult tissues. Elevated survivin levels are found in malignant systemic tumors, and are associated with chemo-resistance, radiation resistance, and poor prognosis. However, expression of survivin in primary nervous system tumors has not been previously characterized. Immunohistochemistry using anti-human survivin antibody (SURV11-A) was performed on formalin-fixed, paraffin-embedded archival tissue from 112 primary central nervous system tumors. Survivin immunoreactivity was seen in most diffuse astrocytomas [WHO II (2/4), III (3/3), IV (9/10), giant-cell glioblastoma (1), and gliosarcoma (1)]. The intensity and degree of survivin expression showed trends with tumor grade, with glioblastomas having the highest positivity. Pilocytic astrocytomas (5) and pleomorphic xanthoastrocytoma (1) were positive to a lesser degree. In oligodendrogliomas (6) and mixed oligo-astrocytomas [grade II (5), II-III (3), and III (7)], oligodendroglial elements appear to be negative compared to positive mini-gemistocytic oligodendrocytes. Ependymomas [grade II (6) and grade III (1)] were positive. Medulloblastomas (5) and retinoblastoma (1/4) showed focal positivity. All meningiomas [grade I (12), II (9), III (4), and grade I (3) and II (5) with frank brain invasion] were intensely positive. All schwannomas (11) and neurofibromas (6) were intensely positive. Thus, survivin is expressed in the majority of the primary nervous system tumors, particularly in glioblastomas, meningiomas, schwannomas and neurofibromas. Overexpression of survivin in meningiomas and benign peripheral nerve sheath tumors contrasts with previous reports relating it to rapid division and poor prognosis.
[Show abstract][Hide abstract] ABSTRACT: Lung resistance-related protein (LRP) was identified as the major vault protein (MVP), the main component of multimeric vault particles. It functions as a transport-associated protein that can be associated with multidrug resistance. In previous studies, expression of MVP/LRP has been documented in tumors of various types. In general, good correlations have been reported for expression of MVP/LRP and decreased sensitivity to chemotherapy and poor prognosis. MVP/LRP expression has been documented in glioblastomas, but its expression in nervous system tumors in general has not been well characterized. Immunohistochemistry using anti-human MVP/LRP antibody (LRP-56) was performed on formalin-fixed, paraffin-embedded archival tissue from 69 primary central nervous system tumors. Expression of MVP/LRP was observed in 81.2% (56/69) of primary nervous system tumors, including astrocytomas (11/13), oligodendrogliomas (1/2), oligoastrocytomas (5/5), ependymoma (1/1), meningiomas (35/45), schwannomas (2/2), and neurofibroma (1/1). Various degrees and distributions of immunoreactivity to MVP/ LRP were observed. Neither the presence nor the degree of immunoreactivity to MVP/LRP showed any correlation with either tumor grade or the presence of brain invasion.